I go A. L. LEHNINGER, G. D. GREVILLE VOL. 12 (1953) 



To prepare extracts the powder was triturated with 10 parts by weight of cold 0.02 M NaHCOg 

 for 2o'-3o' in an ice bath and centrifuged in the cold at 20,000 g for 20 minutes. The clear, reddish 

 supernatant was then exhaustively dialyzed for 4-6 hours in thin cellulose tubing (diameter 0.75 cm), 

 with constant efficient stirring, against several changes of cold 0.02 M NaHCOg. The clear dialyzed 

 extract was again centrifuged at 20,000 g for 20 minutes and the supernatant was then stored in the 

 frozen state at — 15° Cin which form it was stable for at least 4 weeks. Extracts prepared in this manner 

 from KCl-washed particulate material {i.e., the second procedure above) contained little or no /3- 

 hydroxybutyric dehydrogenase, whereas those from mitochondria prepared from sucrose appeared 

 to contain considerable activity. 



Analytical methods. Manometric experiments were carried out employing standard equipment 

 and techniques. Air was used as the gas phase in all the manometric experiments reported. Times 

 quoted in protocols of such experiments were measured from the entry of the vessel into the bath. 

 Taps were closed at five minutes. Oxygen uptakes were corrected for the 5 minute equilibration period 

 by extrapolation. 



Pyruvic acid was used as the lithium salt, other organic acids as K or Na salts. Most of the 

 Coenzyme A used in this work was obtained from Pabst Laboratories, Inc., Milwaukee, Wisconsin 

 and was about 80% pure. The DPN used was 85-90% pure (Sigma Chemical Co., St. Louis). 



Hydroxamic acid formation was measured by a micro modification of the method of Lipmann 

 ."vndTuttle^^. Isolation of hydroxamic acids and their paper chromatography were carried out by the 

 methods described by Stadtman and Barker^^. Whatman No. 43 paper was used most successfully 

 with the butanol-water system. 



Citric acid was determined in trichloracetic acid filtrates by the method of Speck, Moulder and 

 Evans^^. It was found desirable to use a standard batch of ethylene glycol in order to obtain standards 

 of consistent optical density. Colour was allowed to develop for 25 minutes at 20° and read at 457 m//., 

 at which the absorption was found to be maximal, in the Unicam D. G. Spectrophotometer. Micro- 

 determinations of citric acid were performed by the method of Natelson, Lugovoy and Pincus^^. 



Acetoacetate was determined by the method described by Barkulis and Lehninger-^. With 

 British specimens of HCl it was necessary to extract the 2 AT' HCl solution of 2,4-dinitrophenyl- 

 hydrazine several times with CCl j to remove an otherwise very troublesome chromogen. 



EXPERIMENTAL RESULTS 



Previous work^^ has shown that j8-hydroxybiityrate (BOH) is oxidized to aceto- 

 acetate by well-washed preparations of rat Hver mitochondria according to the equation 



/-/3-hydroxybutyrate -f [0] — > acetoacetate + HoO (i) 



presumably by the ^-specific DPN-linked ^-hydroxybutyric dehydrogenase^. Aceto- 

 acetate is apparently inert and accumulates quantitatively in such systems^^-^^. Although 

 oxygen uptake data with rabbit kidney "cyclophorase" preparations indicate that both 

 isomers of the J/-BOH used as substrate undergo oxidation^' ^^, no report has been made 

 of separate tests of the two pure isomers. 



In Table II are shown typical data we have collected on the oxidation of the pure 

 optical isomers of /3-hydroxybutyrate under various conditions by rat liver and kidney 

 mitochondrial preparations. It is seen from experiments i, 2, and 3, Table II, that, as 

 expected, the /-isomer is oxidized to acetoacetate in the absence of fumaratc by liver 

 preparations, the oxygen uptake and acetoacetate formation agreeing fairly well with 

 the stoichiometry expressed by equation (i). Under the same conditions, the ^-isomer 

 is not attacked and no acetoacetate is formed, indicating that the /-specific ^-hydroxy- 

 butyric dehydrogenase is involved in the oxidation of the /-isomer under these particular 

 conditions. 



On the other hand, in the presence of fumarate the ^/-isomer docs undergo oxidation 

 by liver mitochondria as indicated by extra oxygen uptake and the accumulation of 

 extra citrate in large amounts, comparable to the amounts which accumulate when 

 pyruvate is oxidized in the presence of fumarate under the same conditions. It would 

 appear then that the ^/-isomer undergoes oxidation in the presence of an oxalacetate 



References p. 202. 



